Development and characterization of a novel, megakaryocyte NF-κB reporter cell line for investigating inflammatory responses.

Essentials An easily detectable readout in megakaryocyte cell lines will enhance inflammatory research in these cells. Here, we report the development and characterization of a novel megakaryocyte NF-κB-reporter cell line (Meg-01R). Multiple inflammatory molecules modulate NF-κB activity in Meg-01R cells. Meg-01R cells respond to small molecule inhibitors such as IMD0354 and C87 that are known to inhibit NF-κB activity upon stimulation with TNFα. ABSTRACT: Background Because of the difficulties in acquiring large numbers of megakaryocytes, the impact of inflammatory responses on these cells and their ability to produce fully functional platelets under various pathological conditions has not been investigated in detail. Objectives The primary objective of this study is to develop and functionally characterize a novel megakaryocyte nuclear factor κB (NF-κB) reporter cell line to determine the effects of various inflammatory molecules on megakaryocytes and their signalling pathways. Methods A Meg-01-NF-κB-GFP-Luc (Meg-01R) cell line was developed by inserting a reporter NF-κB-GFP-Luc cassette into normal Meg-01 cells to produce luciferase following activation of NF-κB to enable easy detection of pro-inflammatory and reparative signalling. Results and conclusions Meg-01 and Meg-01R cells have comparable characteristics, including the expression of both GPIbα and integrin ß3 . Meg-01R cells responded to various inflammatory molecules as measured by NF-κB-dependent bioluminescence. For example, inflammatory molecules such as tumor necrosis factor-α and Pam3CSK4 increased NF-κB activity, whereas an antimicrobial peptide, LL37, reduced its activity. Meg-01R cells were also found to be sensitive to inhibitors (IMD0354 and C87) of inflammatory pathways. Notably, Meg-01R cells were able to respond to lipopolysaccharide (LPS; non-ultrapure), although it was not able to react to ultrapure LPS because of the lack of sufficient TLR4 molecules on their surface. For the first time, we report the development and characterization of a novel megakaryocyte NF-κB reporter cell line (Meg-01R) as a robust tool to study the inflammatory responses/signalling of megakaryocytes upon stimulation with a broad range of inflammatory molecules that can affect NF-κB activity.

Combined bezafibrate, medroxyprogesterone acetate and valproic acid treatment inhibits osteosarcoma cell growth without adversely affecting normal mesenchymal stem cells.

Drug repurposing is a cost-effective means of targeting new therapies for cancer. We have examined the effects of the repurposed drugs, bezafibrate, medroxyprogesterone acetate and valproic acid on human osteosarcoma cells, i.e., SAOS2 and MG63 compared with their normal cell counterparts, i.e. mesenchymal stem/stromal cells (MSCs). Cell growth, viability and migration were measured by biochemical assay and live cell imaging, whilst levels of lipid-synthesising enzymes were measured by immunoblotting cell extracts. These drug treatments inhibited the growth and survival of SAOS2 and MG63 cells most effectively when used in combination (termed V-BAP). In contrast, V-BAP treated MSCs remained viable with only moderately reduced cell proliferation. V-BAP treatment also inhibited migratory cell phenotypes. MG63 and SAOS2 cells expressed much greater levels of fatty acid synthase and stearoyl CoA desaturase 1 than MSCs, but these elevated enzyme levels significantly decreased in the V-BAP treated osteosarcoma cells prior to cell death. Hence, we have identified a repurposed drug combination that selectively inhibits the growth and survival of human osteosarcoma cells in association with altered lipid metabolism without adversely affecting their non-transformed cell counterparts.

Optically Transparent Anionic Nanofibrillar Cellulose Is Cytocompatible with Human Adipose Tissue-Derived Stem Cells and Allows Simple Imaging in 3D.

The anti-inflammatory and immunomodulatory properties of human mesenchymal stromal cells (MSCs) are a focus within regenerative medicine. However, 2D cultivation of MSCs for extended periods results in abnormal cell polarity, chromosomal changes, reduction in viability, and altered differentiation potential. As an alternative, various 3D hydrogels have been developed which mimic the endogenous niche of MSCs. Nevertheless, imaging cells embedded within 3D hydrogels often suffers from low signal-to-noise ratios which can be at least partly attributed to the high light absorbance and light scattering of the hydrogels in the visible light spectrum. In this study, human adipose tissue-derived MSCs (ADSCs) are cultivated within an anionic nanofibrillar cellulose (aNFC) hydrogel. It is demonstrated that aNFC forms nanofibres arranged as a porous network with low light absorbance in the visible spectrum. Moreover, it is shown that aNFC is cytocompatible, allowing for MSC proliferation, maintaining cell viability and multilineage differentiation potential. Finally, aNFC is compatible with scanning electron microscopy (SEM) and light microscopy including the application of conventional dyes, fluorescent probes, indirect immunocytochemistry, and calcium imaging. Overall, the results indicate that aNFC represents a promising 3D material for the expansion of MSCs whilst allowing detailed examination of cell morphology and cellular behaviour.